Expression of Smyd1b_tv1 by Alternative Splicing in Cardiac Muscle is Critical for Sarcomere Organization in Cardiomyocytes and Heart Function.

Smyd1, a member of the Smyd lysine methyltransferase family, plays an important role in myofibrillogenesis of skeletal and cardiac muscles. Loss of Smyd1b (a Smyd1 ortholog) function in zebrafish results in embryonic death from heart malfunction. smyd1b encodes two isoforms, Smyd1b_tv1 and Smyd1b_tv2, differing by 13 amino acids due to alternative splicing. While smyd1 alternative splicing is evolutionarily conserved, the isoform-specific expression and function of Smyd1b_tv1 and Smyd1b_tv2 remained unknown. Here we analyzed their expression and function in skeletal and cardiac muscles. Our analysis revealed expression of smyd1b_tv1 predominately in cardiac and smyd1b_tv2 in skeletal muscles. Using zebrafish models expressing only one isoform, we demonstrated that Smyd1b_tv1 is essential for cardiomyocyte differentiation and fish viability, whereas Smyd1b_tv2 is dispensable for heart development and fish survival. Cellular and biochemical analyses revealed that Smyd1b_tv1 differs from Smyd1b_tv2 in protein localization and binding with myosin chaperones. While Smyd1b_tv2 diffused in the cytosol of muscle cells, Smyd1b_tv1 was localized to M-lines and essential for sarcomere organization in cardiomyocytes. Co-IP analysis revealed a stronger binding of Smyd1b_tv1 with chaperones and cochaperones compared with Smyd1b_tv2. Collectively, these findings highlight the nonequivalence of Smyd1b isoforms in cardiomyocyte differentiation, emphasizing the critical role of Smyd1b_tv1 in cardiac function.

Examination of wnt signaling mediated melanin transport and shell color formation in Pacific oyster (Crassostrea gigas).

Mollusca exhibit remarkable diversity in shell coloration, attributed to the presence of melanin, a widely distributed pigment with various essential roles, such as mechanical strengthening, antioxidation and thermoregulation. However, the regulatory network governing melanogenesis and melanin transport in molluscs remains poorly understood. In this study, we conducted a systematic analysis of melanin distribution and transport in the Pacific oyster, utilizing light microscopy and high-resolution transmission electron microscopy. In addition, we characterized CgWnt1 and CgWnt2b-a in Crassostrea gigas, and analyzed Wnt signaling in melanocyte formation. Expression analysis revealed that these genes were predominantly expressed in the mantle of black-shelled individuals, particularly in the outer fold of the mantle. Furthermore, we employed RNA interference and inhibitors to specifically inhibit Wnt signaling in both in vivo and in vitro. The results revealed impaired melanogenesis and diminished tyrosinase activity upon Wnt signaling inhibition. These findings suggest the crucial role of Wnt ligands and downstream factors in melanogenesis. In summary, our study provides valuable insights into the regulatory mechanism of shell pigmentation in C. gigas. By demonstrating the promotion of melanogenesis through Wnt signaling modulation, we contribute to a better understanding of the complex processes underlying molluscan melanin production and shell coloration. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00221-5.

Ectopic expression of Myomaker and Myomixer in slow muscle cells induces slow muscle fusion and myofiber death.

Zebrafish embryos possess two major types of myofibers, the slow and fast fibers, with distinct patterns of cell fusion. The fast muscle cells can fuse, while the slow muscle cells cannot. Here, we show that myomaker is expressed in both slow and fast muscle precursors, while myomixer is exclusive to fast muscle cells. The loss of Prdm1a, a regulator of slow muscle differentiation, results in strong myomaker and myomixer expression and slow muscle cell fusion. This abnormal fusion is further confirmed by the direct ectopic expression of myomaker or myomixer in slow muscle cells of transgenic models. Using the transgenic models, we show that the heterologous fusion between slow and fast muscle cells can alter slow muscle cell migration and gene expression. Furthermore, the overexpression of myomaker and myomixer also disrupts membrane integrity, resulting in muscle cell death. Collectively, this study identifies that the fiber-type-specific expression of fusogenic proteins is critical for preventing inappropriate fusion between slow and fast fibers in fish embryos, highlighting the need for precise regulation of fusogenic gene expression to maintain muscle fiber integrity and specificity.

Protoporphyrin IX metabolism mediated via translocator protein (CgTspO) involved in orange shell coloration of pacific oyster (Crassostrea gigas).

Mollusc shell color polymorphism is influenced by various factors. Pigments secreted in vivo by animals play a critical role in shell coloration. Among the different shell-color hues, orange pigmentation has been partially attributed to porphyrins. However, the detailed causal relationship between porphyrins and orange-shell phenotype in molluscs remains largely unexplored. The various strains of Pacific oyster (Crassostrea gigas) with different shell color provide useful models to study the molecular regulation of mollusc coloration. Accordingly, oysters with orange and gold-shells, exhibiting distinct porphyrin distributions, were selected for analysis of total metabolites and gene expression profile through mantle metabolomic and transcriptomic studies. Translocator protein (TspO) and protoporphyrin IX (PPIX) were identified as potential factors influencing oyster shell-color. The concentration of PPIX was measured using HPLC, while expression profiling of CgTspO was analyzed by qPCR, in situ hybridization, Western blotting, and immunofluorescence techniques. Moreover, the roles of CgTspO in regulating PPIX metabolism and affecting the orange-shell-coloration were investigated in vitro and in vivo. These studies indicate that PPIX and its associated metabolic protein, CgTspO may serve as new regulators of orange-shell-coloration in C. gigas. Data of this study offer new insights into oyster shell coloration and enhancing understandings of mollusc shell color polymorphism.

OysterDB: A Genome Database for Ostreidae.

The molluscan family Ostreidae, commonly known as oysters, is an important molluscan group due to its economic and ecological importance. In recent years, an abundance of genomic data of Ostreidae species has been generated and available in public domain. However, there is still a lack of a high-efficiency database platform to store and distribute these data with comprehensive tools. In this study, we developed an oyster genome database (OysterDB) to consolidate oyster genomic data. This database includes eight oyster genomes and 208,923 protein-coding gene annotations. Bioinformatic tools, such as BLAST and JBrowse, are integrated into the database to provide a user-friendly platform for homologous sequence searching, visualization of genomes, and screen for candidate gene information. Moreover, OysterDB will be continuously updated with ever-growing oyster genomic resources and facilitate future studies for comparative and functional genomic analysis of oysters ( http://oysterdb.com.cn/ ).

Alternative splicing of histone demethylase Kdm6bb mediates temperature-induced sex reversal in the Nile tilapia.

Sex determination in many fish species is remarkably plastic and temperature sensitive. Nile tilapia display a genetic sex-determination system (XX/XY). However, high-temperature treatment during critical thermosensitive periods can induce XX females into XXm pseudo-males, and this phenomenon is termed temperature-induced sex reversal (TISR). To investigate the molecular mechanism of TISR in Nile tilapia, we performed Iso-seq analysis and found a dramatic effect of high temperature on gene alternative splicing (AS). Kdm6bb histone demethylase showed a novel AS at intron 5 that generates Kdm6bb_tv1 transcripts without intron 5 and Kdm6bb_tv2 with intron 5. Kdm6bb_tv1 encodes a full-length protein while Kdm6bb_tv2 encodes a truncated protein. Expression analysis revealed that intron 5 splicing of Kdm6bb is male and gonad biased at larval stage, and only gonad biased at adult stage. High-temperature treatment induced intron 5 splicing in the gonads of XX and XY fish, resulting in increased Kdm6bb_tv1 expression. To directly test the role of Kdm6bb_tv1 in Nile tilapia TISR, we knocked out expression of Kdm6bb_tv1. However, Kdm6bb_tv1-/- homozygous mutants showed embryonic lethality. Overexpression of Kdm6bb_tv1, but not Kdm6bb_tv2, induced sex reversal of XX females into pseudo-males. Overexpression of Kdm6bb_tv1, as with high-temperature treatment, modified the promotor region of Gsdf and Dmrt1 by demethylating the trimethylated lysine 27 of histone 3 (H3K27me3), thereby increasing expression. Collectively, these studies demonstrate that AS of Kdm6bb intron 5 increases the expression of Kdm6bb_tv1, which acts as a direct link between high temperature and activation of Gsdf and Dmrt1 expression, leading to male sex determination.

Chromosome-level genome assembly of sea scallop Placopecten magellanicus provides insights into the genetic characteristics and adaptive evolution of large scallops.

Placopecten magellanicus (Gmelin, 1791), a deep-sea Atlantic scallop, holds significant commercial value as a benthic marine bivalve along the northwest Atlantic coast. Recognizing its economic importance, the need to reconstruct its genome assembly becomes apparent, fostering insights into natural resources and generic breeding potential. This study reports a high-quality chromosome-level genome of P. magellanicus, achieved through the integration of Illumina short read sequencing, PacBio HiFi sequencing, and Hi-C sequencing techniques. The resulting assembly spans 1778 Mb with a scaffold N50 of 86.71 Mb. An intriguing observation arises - the genome size of P. magellanicus surpasses that of its Pectinidae family peers by 1.80 to 2.46 times. Within this genome, 28,111 protein-coding genes were identified. Comparative genomic analysis involving five scallop species unveils the critical determinant of this expanded genome: the proliferation of repetitive sequences recently inserted, contributing to its enlarged size. The landscape of whole genome collinearity sheds light on the relationships among scallop species, enhancing our broader understanding of their genomic framework. This genome provides genomic resources for future molecular biology research on scallops and serves as a guide for the exploration of longevity-related genes in scallops.

A chromosome-level genome assembly of Ostrea denselamellosa provides initial insights into its evolution.

The oyster Ostrea denselamellosa is a live-bearing species with a sharp decline in the natural population. Despite recent breakthroughs in long-read sequencing, high quality genomic data are very limited in O. denselamellosa. Here, we carried out the first whole genome sequencing at the chromosome-level in O. denselamellosa. Our studies yielded a 636 Mb assembly with scaffold N50 around 71.80 Mb. 608.3 Mb (95.6% of the assembly) were anchored to 10 chromosomes. A total of 26,412 protein-coding genes were predicted, of which 22,636 (85.7%) were functionally annotated. By comparative genomics, we found that long interspersed nuclear element (LINE) and short interspersed nuclear element (SINE) made up a larger proportion in O. denselamellosa genome than in other oysters'. Moreover, gene family analysis showed some initial insight into its evolution. This high-quality genome of O. denselamellosa provides a valuable genomic resource for studies of evolution, adaption and conservation in oysters.

Loss of Myomixer Results in Defective Myoblast Fusion, Impaired Muscle Growth, and Severe Myopathy in Zebrafish.

The development and growth of fish skeletal muscles require myoblast fusion to generate multinucleated myofibers. While zebrafish fast-twitch muscle can fuse to generate multinucleated fibers, the slow-twitch muscle fibers remain mononucleated in zebrafish embryos and larvae. The mechanism underlying the fiber-type-specific control of fusion remains elusive. Recent genetic studies using mice identified a long-sought fusion factor named Myomixer. To understand whether Myomixer is involved in the fiber-type specific fusion, we analyzed the transcriptional regulation of myomixer expression and characterized the muscle growth phenotype upon genetic deletion of myomixer in zebrafish. The data revealed that overexpression of Sonic Hedgehog (Shh) drastically inhibited myomixer expression and blocked myoblast fusion, recapitulating the phenotype upon direct genetic deletion of myomixer from zebrafish. The fusion defect in myomixer mutant embryos could be faithfully rescued upon re-expression of zebrafish myomixer gene or its orthologs from shark or human. Interestingly, myomixer mutant fish survived to adult stage though were notably smaller than wildtype siblings. Severe myopathy accompanied by the uncontrolled adipose infiltration was observed in both fast and slow muscle tissues of adult myomixer mutants. Collectively, our data highlight an indispensable role of myomixer gene for cell fusion during both embryonic muscle development and post-larval muscle growth.

A high-quality chromosome-level genome assembly of the bivalve mollusk Mactra veneriformis.

Mactra veneriformis (Bivalvia: Mactridae) is a bivalve mollusk of major economic importance in China. Decreased natural yields of M. veneriformis have led to an urgent need for genomic resources. To address this problem and the currently limited knowledge of molecular evolution in this genus, we here report a high-quality chromosome-level genome assembly of M. veneriformis. Our approach yielded a 939.32 Mb assembled genome with an N50 contig length of 7,977.84 kb. Hi-C scaffolding of the genome resulted in assembly of 19 pseudochromosomes. Repetitive elements made up ∼51.79% of the genome assembly. A total of 29,315 protein-coding genes (PCGs) were predicted in M. veneriformis. Construction of a genome-level phylogenetic tree demonstrated that M. veneriformis and Ruditapes philippinarum diverged around 231 million years ago (MYA). Inter-species comparisons revealed that 493 gene families have undergone expansion and 449 have undergone contraction in the M. veneriformis genome. Chromosome-based macrosynteny analysis revealed a high degree of synteny between the 19 chromosomes of M. veneriformis and those of Patinopecten yessoensis. These results suggested that M. veneriformis has a similar karyotype to that of P. yessoensis, and that a highly conserved 19-chromosome karyotype was formed in the early differentiation stages of bivalves. In summary, the genomic resources generated in this work serve as a valuable reference for investigating the molecular mechanisms underlying biological functions in M. veneriformis and will facilitate future genetic improvement and disease treatment in this economically important species. Furthermore, the assembled genome greatly improves our understanding of early genomic evolution of the Bivalvia.

Gene expression and functional analysis of Aha1a and Aha1b in stress response in zebrafish.

Activator of heat shock protein 90 (hsp90) ATPase (Aha1) is a Hsp90 co-chaperone required for Hsp90 ATPase activation. Aha1 is essential for yeast survival and muscle development in C. elegans under elevated temperature and hsp90-deficeiency induced stress conditions. The roles of Aha1 in vertebrates are poorly understood. Here, we characterized the expression and function of Aha1 in zebrafish. We showed that zebrafish genome contains two aha1 genes, aha1a and aha1b, that show distinct patterns of expression during development. Under the normal physiological conditions, aha1a is primarily expressed in skeletal muscle cells of zebrafish embryos, while aha1b is strongly expressed in the head region. aha1a and aha1b expression increased dramatically in response to heat shock induced stress. In addition, Aha1a-GFP fusion protein exhibited a dynamic translocation in muscle cells in response to heat shock. Moreover, upregulation of aha1 expression was also observed in hsp90a1 knockdown embryos that showed a muscle defect. Genetic studies demonstrated that knockout of aha1a, aha1b or both had no detectable effect on embryonic development, survival, and growth in zebrafish. The aha1a and aha1b mutant embryos showed normal muscle development and stress response in response to heat shock. Single or double aha1a and aha1b mutants could grow into normal reproductive adults with normal skeletal muscle structure and morphology compared with wild type control. Together, data from these studies indicate that Aha1a and Aha1b are involved in stress response. However, they are dispensable in zebrafish embryonic development, growth, and survival.

The cellular architecture and molecular determinants of the zebrafish fusogenic synapse.

Myoblast fusion is an indispensable process in skeletal muscle development and regeneration. Studies in Drosophila led to the discovery of the asymmetric fusogenic synapse, in which one cell invades its fusion partner with actin-propelled membrane protrusions to promote fusion. However, the timing and sites of vertebrate myoblast fusion remain elusive. Here, we show that fusion between zebrafish fast muscle cells is mediated by an F-actin-enriched invasive structure. Two cell adhesion molecules, Jam2a and Jam3b, are associated with the actin structure, with Jam2a being the major organizer. The Arp2/3 actin nucleation-promoting factors, WAVE and WASP-but not the bipartite fusogenic proteins, Myomaker or Myomixer-promote the formation of the invasive structure. Moreover, the convergence of fusogen-containing microdomains and the invasive protrusions is a prerequisite for cell membrane fusion. Thus, our study provides unprecedented insights into the cellular architecture and molecular determinants of the asymmetric fusogenic synapse in an intact vertebrate animal.

XBP1 variant 1 promotes mitosis of cancer cells involving upregulation of the polyglutamylase TTLL6.

XBP1 variant 1 (Xv1) is the most abundant XBP1 variant and is highly enriched across cancer types but nearly none in normal tissues. Its expression is associated with poor patients' survival and is specifically required for survival of malignant cells, but the underlying mechanism is not known. Here we report that Xv1 upregulates the polyglutamylase tubulin tyrosine ligase-like 6 (TTLL6) and promotes mitosis of cancer cells. Like the canonical XBP1, Xv1 mRNA undergoes unconventional splicing by IRE1α under endoplasmic reticulum stress, but it is also constitutively spliced by IRE1ß. The spliced Xv1 mRNA encodes the active form of Xv1 protein (Xv1s). RNA sequencing in HeLa cells revealed that Xv1s overexpression regulates expression of genes that are not involved in the canonical unfolded protein response, including TTLL6 as a highly upregulated gene. Gel shift assay and chromatin immunoprecipitation revealed that Xv1s bind to the TTLL6 promoter region. Knockdown of TTLL6 caused death of cancer cells but not benign and normal cells, similar to the effects of knocking down Xv1. Moreover, overexpression of TTLL6 partially rescued BT474 cells from apoptosis induced by either TTLL6 or Xv1 knockdown, supporting TTLL6 as an essential downstream effector of Xv1 in regulating cancer cell survival. TTLL6 is localized in the mitotic spindle of cancer cells. Xv1 or TTLL6 knockdown resulted in decreased spindle polyglutamylation and interpolar spindle, as well as congression failure, mitotic arrest and cell death. These findings suggest that Xv1 is essential for cancer cell mitosis, which is mediated, at least in part, by increasing TTLL6 expression.

Wide temperature operation of kilowatt fiber oscillators.

To meet the requirements of fiber laser applications under extreme temperatures or when there is a large temperature difference, it is necessary to develop fiber lasers able to operate in a wide temperature range. At present, there is a lack of reports on high-power fiber lasers that can operate in a wide temperature range with low power fluctuations. Thus, we designed a 1 kW fiber oscillator that can operate in a wide temperature range through temperature-related rate equations. The output characteristics of the oscillator are measured in the operating temperature range from -30∘C to 20°C. The experimental results show that the laser output power fluctuates by 7% over the entire temperature range. It was discovered that as the ambient temperature decreased, the efficiency of the laser decreased, and this issue is discussed in detail. This work has guiding significance for the design of high-power fiber lasers operating at a wide temperature range, and simultaneously, to the best of our knowledge, it provides the first kilowatt fiber oscillator that can operate in a wide temperature range between -30∘C and 20°C.

Chromosome-level genome assembly of Scapharca kagoshimensis reveals the expanded molecular basis of heme biosynthesis in ark shells.

Ark shells are commercially important clam species that inhabit in muddy sediments of shallow coasts in East Asia. For a long time, the lack of genome resources has hindered scientific research of ark shells. Here, we report a high-quality chromosome-level genome assembly of Scapharca kagoshimensis, with an aim to unravel the molecular basis of heme biosynthesis, and develop genomic resources for genetic breeding and population genetics in ark shells. Nineteen scaffolds corresponding to 19 chromosomes were constructed from 938 contigs (contig N50 = 2.01 Mb) to produce a final high-quality assembly with a total length of 1.11 Gb and scaffold N50 around 60.64 Mb. The genome assembly represents 93.4% completeness via matching 303 eukaryota core conserved genes. A total of 24,908 protein-coding genes were predicted and 24,551 genes (98.56%) of which were functionally annotated. The enrichment analyses suggested that genes in heme biosynthesis pathways were expanded and positive selection of the haemoglobin genes was also found in the genome of S. kagoshimensis, which gives important insights into the molecular mechanisms and evolution of the heme biosynthesis in mollusca. The valuable genome assembly of S. kagoshimensis would provide a solid foundation for investigating the molecular mechanisms that underlie the diverse biological functions and evolutionary adaptations of S. kagoshimensis.

Overexpression of Lifeact-GFP Disrupts F-Actin Organization in Cardiomyocytes and Impairs Cardiac Function.

Lifeact-GFP is a frequently used molecular probe to study F-actin structure and dynamic assembly in living cells. In this study, we generated transgenic zebrafish models expressing Lifeact-GFP specifically in cardiac muscles to investigate the effect of Lifeact-GFP on heart development and its application to study cardiomyopathy. The data showed that transgenic zebrafish with low to moderate levels of Lifeact-GFP expression could be used as a good model to study contractile dynamics of actin filaments in cardiac muscles in vivo. Using this model, we demonstrated that loss of Smyd1b, a lysine methyltransferase, disrupted F-actin filament organization in cardiomyocytes of zebrafish embryos. Our studies, however, also demonstrated that strong Lifeact-GFP expression in cardiomyocytes was detrimental to actin filament organization in cardiomyocytes that led to pericardial edema and early embryonic lethality of zebrafish embryos. Collectively, these data suggest that although Lifeact-GFP is a good probe for visualizing F-actin dynamics, transgenic models need to be carefully evaluated to avoid artifacts induced by Lifeact-GFP overexpression.

Smyd1 is essential for myosin expression and sarcomere organization in craniofacial, extraocular, and cardiac muscles.

Skeletal and cardiac muscles are striated myofibers that contain highly organized sarcomeres for muscle contraction. Recent studies revealed that Smyd1, a lysine methyltransferase, plays a key role in sarcomere assembly in heart and trunk skeletal muscles. However, Smyd1 expression and function in craniofacial muscles are not known. Here, we analyze the developmental expression and function of two smyd1 paralogous genes, smyd1a and smyd1b, in craniofacial and cardiac muscles of zebrafish embryos. Our data show that loss of smyd1a (smyd1amb5) or smyd1b (smyd1bsa15678) has no visible effects on myogenic commitment and expression of myod and myosin heavy-chain mRNA transcripts in craniofacial muscles. However, myosin heavy-chain protein accumulation and sarcomere organization are dramatically reduced in smyd1bsa15678 single mutant, and almost completely diminish in smyd1amb5; smyd1bsa15678 double mutant, but not in smyd1amb5 mutant. Similar defects are also observed in cardiac muscles of smyd1bsa15678 mutant. Defective craniofacial and cardiac muscle formation is associated with an upregulation of hsp90α1 and unc45b mRNA expression in smyd1bsa15678 and smyd1amb5; smyd1bsa15678 mutants. Together, our studies indicate that Smyd1b, but not Smyd1a, plays a key role in myosin heavy-chain protein expression and sarcomere organization in craniofacial and cardiac muscles. Loss of smyd1b results in muscle-specific stress response.

A novel XBP1 variant is highly enriched in cancer tissues and is specifically required for cancer cell survival.

XBP1 is a basic leucine zipper (bZIP) transcription factor and a key mediator of the endoplasmic reticulum (ER) stress-activated unfolded protein response (UPR). XBP1-mediated transcription facilitates cell adaptation to ER stress and also promotes tumor progression, while suppressing anti-tumor immunity. Here we report a novel XBP1 variant, namely XBP1 variant 1 (XBP1v1, Xv1 for short), that is specifically required for survival of cancer cells. Xv1 contains a cryptic first exon that is conserved only in humans and great apes. Comparing to XBP1, Xv1 encodes a protein with a different N-terminal sequence containing 25 amino acids. Analysis of RNAseq database reveals that Xv1 is broadly expressed across cancer types but almost none in normal tissues. Elevated Xv1 expression is associated with poor survival of patients with several types of cancer. Knockdown of Xv1 induces death of multiple cancer cell lines but has little effect on non-cancerous cells in vitro. Moreover, knockdown of Xv1 also inhibits growth of a xenograft breast tumor in mice. Together, our results indicate that Xv1 is essential for survival of cancer cells.

Characterization of paramyosin protein structure and gene expression during myogenesis in Pacific oyster (Crassostrea gigas).

Paramyosin is a key component of thick filaments in invertebrate muscles. In this study, we isolated the full length cDNA of paramyosin from Pacific oyster (Crassostrea gigas), and determined its pattern of expression during myogenesis. The full length paramyosin (CgPM) cDNA contains an open reading frame (ORF) of 2586 bp encoding a 861-amino acid protein. Sequence analysis revealed an assembly competence domain (ACD) and a heptad repeat (d-e-f-g-a-b-c) with 28-residue repeat zones in the CgPM primary structure, a characteristic of coiled-coil protein. Quantitative analysis of CgPM expression revealed a sharp increase in trochophore stage, and peaked at the D-shaped stage. Strong CgPM expression was found in smooth adductor muscle, followed by striated adductor muscle and mantle tissue. Whole-mount in situ hybridization (WISH) showed a restricted pattern of CgPM expression in adductor muscle, larval velum retractor and foot muscles at the umbo and eyed larval stages. These data indicate that CgPM is strongly expressed during larval myogenesis in C. gigas, which provides the basis for further functional studies of paramyosin in oyster to better understand the molecular and cellular mechanisms of muscle formation in mollusks.

Identification and characterization of key haem pathway genes associated with the synthesis of porphyrin in Pacific oyster (Crassostrea gigas).

Molluscs exhibit diverse shell colors. The molecular regulation of shell coloration is however not well understood. To investigate the connection of shell coloration with pigment synthesis, we analyzed the distribution of porphyrins, a widespread group of pigments in nature, in four Pacific oyster strains of different shell colors including black, orange, golden, and white. The porphyrin distribution was analyzed in oyster mantles and shells by fluorescence imaging and UV spectrophotometer. The results showed that red fluorescence emitted by porphyrins under the UV light was detected only on the nacre of the orange-shell strain and mantles of orange, black and white-shell strains. Extracts from newly deposit shell, nacre and mantle tissue from orange-shell specimens showed peaks in UV-vis spectra that are characteristic of porphyrins, but these were not observed for the other shell-color strains. In addition, genes of the haem synthetic pathway were isolated and characterized. Phylogenetic analysis of CgALAS, CgALAD, CgPBGD, CgUROS, and CgUROD provide further evidence for a conserved genetic pathway of haem synthesis during evolution. Differential expression of the haem genes expressed in mantle tissues support these findings and are consistent with porphyrins being produced by the orange strain only. Tissue in situ hybridization demonstrated the expression of these candidate genes at the outer fold of C. gigas mantles where shell is deposited. Our studies provide a better understanding of shell pigmentation in C. gigas and candidate genes for future mechanistic analysis of shell color formation in molluscs.